The anticonvulsant BW534U87 depresses epileptiform activity in rat hippocampal slices by an adenosine-dependent mechanism and through inhibition of voltage-gated Na+ channels

Abstract

1. The cellular and molecular actions of BW534U87 were studied using intracellular and extracellular recordings from the CA1 region of rat hippocampal slices and whole-cell voltage-clamp recordings of recombinant human brain type IIA Na+ channels expressed in Chinese hamster ovary (CHO) cells. 2. Normal excitatory and inhibitory postsynaptic potentials evoked in hippocampal slices were unaffected by BW534U87 or the adenosine deaminase inhibitor EHNA. However, epileptiform activity was depressed by BW534U87 (50 micronM) and this inhibition was reversed by the adenosine receptor antagonist 8-phenyl theophylline (8-PT, 30 micronM). EHNA (10 micronM) mimicked the effects of BW534U87. Furthermore, BW534U87 enhanced the inhibitory effects of exogenous adenosine on evoked synaptic potentials. BW534U87 (50 micronM) also voltage- and use-dependently inhibited action potentials elicited by current injection, independent of the adenosine system, since it was not affected by 8-PT. 3. In CHO cells expressing the recombinant human brain Na+ channel, BW534U87 produced a concentration- and voltage-dependent inhibition of Na+ currents with a half-maximal inhibitory concentration of 10 micronM at a Vh of -60 mV. Use-dependent inhibition was evident at high-frequencies (20x20 ms pulse train at 10 Hz). 4 In conclusion, BW534U87 blocks hippocampal epileptiform activity by a dual mechanism. The first action is similar to that produced by EHNA and is dependent on endogenous adenosine probably by inhibition of adenosine deaminase. Secondly, BW534U87 directly inhibits voltage-gated Na+ channels in a voltage- and frequency-dependent manner. Both actions of BW534U87 are activity-dependent and may synergistically contribute to its overall anticonvulsant effects in animal models of epilepsy.

Figure 1

BW534U87 reduced epileptiform activity in hippocampal slices. (A) Normal excitatory and inhibitory postsynaptic potentials (EPSP–IPSPs) recorded in a CA1 pyramidal cell using a sharp microelectrode containing 4 M K-acetate, evoked by stimulation of the Schaffer collateral/commisural fibres, were not affected by BW534U87 (50 μM). (B) In the same neurone, 50 μM BW534U87 depressed epileptiform activity induced by perfusing the slice with a Mg²⁺-free solution containing 7 mM K⁺ and 20 μM bicuculline. (C) Depression of epileptiform activity recorded with an electrode containing 3 M KCl in a different neurone by 10 μM of the adenosine deaminase inhibitor EHNA. (D) In another neurone, the inhibition of the epileptiform EPSP area was reversed by co-application with the adenosine receptor antagonist 8-phenyl theophylline (8-PT, 30 μM). The membrane potentials of all cells were held at −70 mV.

Figure 2

BW534U87 inhibits spontaneous epileptiform activity in hippocampal slices. Extracellular recording using a glass microelectrode from the CA1 region in a hippocampal slice. Spontaneous population bursts were recorded within 15 min following application of a Mg²⁺-free solution containing 7 mM K+ and 20 μM bicuculline. The top trace shows spontaneous population bursts in the absence of drug (left trace) and the average of 20 population bursts (right trace). Middle traces show the reduction in burst amplitude 15 min following application of 50 μM BW534U87. Bottom traces show the complete reversal of the effects of the drug within 30 min following washout.

Figure 3

BW534U87 and EHNA enhanced the inhibitory effects of exogenous adenosine on evoked synaptic potentials. (A) Inhibition of the EPSP area in a CA1 pryramidal neurone by increasing concentration of exogenously applied adenosine (1–300 μM alone or in the presence of 50 μM BW534U87. (B) Inhibition of the EPSP by adenosine alone or in the presence of 10 μM EHNA. (C) Concentration-response curves for adenosine in the absence of drug (pooled data from nine cells) and in presence of BW534U87 (n= 5 cells) or 10 μM EHNA (n=4 cells). The smooth curves were obtained by fitting the data to an independent-binding-site receptor model. The IC₅₀ for inhibition of the EPSP area by adenosine was estimated at 41.0 μM [29.8–53.9 μM) and a Hill coefficient was 1.8±0.1. In the presence of BW534U87 (50 μM), the IC₅₀ for adenosine was 15.9 μM [7.3–25.8 μM] and the Hill coefficient was 1.5±0.1. In the presence of EHNA (10 μM) the IC₅₀ for adenosine was 18.9 μM [11.7–30.6 μM] and the Hill coefficient was 1.5±0.2.

Figure 4

BW534U87 inhibited the latter action potentials in a train in hippocampal CA1 pyramidal neurones. (A) Trains of action potentials were elicited by a depolarizing current injection (0.8 nA, 600 ms) in normal solution (resting potential −63 mV). At a holding potential of −70 mV (left) BW534U87 (50 μM) inhibited the latter action potentials in the train. At the more hyperpolarized potentials of −80 mV (centre) and −90 mV (right) the effect of BW534U87 was less marked. (B) In another neurone, the inhibition of action potentials by BW534U87 was not affected by co-application with 8-phenyl theophylline (8-PT, 30 μM). (C) Summary of the average number of spikes elicited by depolarizing current injection (0.8 nA, 600 ms) into CA1 pyramidal neurones (n=5 cells) in control, in the presence of 50 μM BW534U87, 30 μM 8-PT or combination of both BW534U87 and 8-PT and after washout of drugs (horizontal hatched column). Values are the mean±s.e. mean. (D) Reduction in the number of action potentials in a train by BW534U87 (50 μM) was dependent on the initial adapted spike frequency prior to drug addition. The Spearman's product moment coefficient was 0.76.

Figure 5

BW534U87 produced a state-dependent inhibition of recombinant human brain Na⁺ channels expressed in Chinese hamster ovary cells under voltage-clamp conditions. (A, upper traces) Currents evoked from a holding potential (V_h) of −90 mV to a range of potentials between −80 and +70 mV in control and following application of 10 μM BW534U87. (Lower traces) Peak current-voltage relationship in control and after application of BW534U87 (five cells). (B, Upper traces) Currents evoked at a test potential of 0 mV (3 ms) following a conditioning pulse of −70 or −60 mV (1 s) in control and in 10 μM BW534U87. (Lower traces) Effect of BW534U87 on the voltage-dependence of inactivation. Channels were inactivated by a range of conditioning pulses between −120 to −120 mV (1 s) prior to stepping to a test potential of 0 mV. Data are normalized to the current generated following a −120 mV conditioning pulse. Data were fitted with a Boltzmann function, where in control, the V_½; was −62.1±1.8 mV and k was 4.4±0.2 mV/e-fold change. In the presence of 10 μM BW534U87, the V_½; was −70.3±1.9 mV and k was 4.6±0.2 mV/e-fold change. (C, Upper traces) Currents evoked by a test pulse to 0 mV (8 ms) from a V_h of −90 or −60 mV (conditioning pulse to −60 mV for 1 s) in control and in 10 μM BW534U87. (Lower traces) Concentration-dependence of inhibition of currents by BW534U87 evoked from a V_h of −90 or −60 mV. The data are normalized with respect to the current amplitudes in the absence of the compound. Each point is the mean±s.e. mean (n=4∼7 cells). The smooth curve was obtained by fitting the data to an independent-binding-site receptor model, from which a half-maximal inhibitory concentration of 10 μM and a Hill coefficient of approximately 1 were estimated. (D) Frequency-dependent inhibition of Na⁺ currents. Currents were elicited by a train of 20 pulses (3.5 ms duration; 10 Hz) from a V_h of −90 to 0 mV. (Upper traces) Currents evoked by the 1st and 20th pulse in the absence of drug and in presence of 10 μM BW534U87. (Lower traces) Average current amplitude (normalized to the first pulse) during a train of 20 pulses in control and in the presence of 10 μM BW534U87. Data points are the mean±s.e. mean (n=6 cells). Leak currents were not subtracted in these experiments.